“Bacteria in milk (not just an-udder microbiology lab)!” Science in the Real World Microbes In Action “Bacteria in milk (not just an-udder microbiology lab)!” is a curriculum unit developed as part of the Science In The Real World: Microbes In Action Program. The curriculum units were developed with support from the National Science Foundation, The Coordinating Board for Higher Education, Sigma Chemical Company, Pfizer Foundation and the Foundation for Microbiology. Don Cohn and Elmer Kellmann Developer of Curriculum Unit Teresa Thiel, Ph. D. Victoria L. May, M.A.T. University of Missouri- St. Louis Science Education Resource Center Program Director & Microbiologist Co- Director & Curriculum Specialist Mark R. Kalk, M.S. Sandra Alters, Ph. D. Science Education Resource Center Brian Alters, Ph. D. Lab Supervisor & Technical Specialist Program Evaluators Kimber Mallet Judith O’ Brien, Ph. D. Illustrator Ralston Purina Industrial Consultant Bruce C. Hemming, Ph. D. Alastair Pringle, Ph. D. Sigma Chemical Company Anheuser- Busch Industrial Consultant Industrial Consultant Robert Reynolds, Ph. D David Corbin, Ph. D. Sigma Chemical Company Monsanto Industrial Consultant Industrial Consultant Copyright © 2003 by University of Missouri-St. Louis All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, recording, or otherwise, without the prior written permission of the copyright owner (except as noted below*). *Teachers may copy only the student pages for educational use. 2003 Science in the Real World: Microbes in Action 1 This material may be duplicated by teachers for use in the classroom. Any other use is prohibited. TABLE OF CONTENTS BACTERIA IN MILK - NOT JUST AN-UDDER MICROBIOLOGY LAB At a Glance Description 3 Time Requirements 3 Curriculum Placement 3 Equipment 3 Materials 3 Timeline 4 Preparations Materials 4 Preparing Milk 4 Preparing Nutrient Agar 5 Sample Preparation 5 Day One 6 Day Two 7 Sources of Supplies 9 Teacher Background 9 Teacher Hints & Troubleshooting 10 Answer Key 10 Student Guide Student Background 13 Student Procedures 14-15 Student Results and Analysis Worksheets 16-17 2003 Science in the Real World: Microbes in Action 2 This material may be duplicated by teachers for use in the classroom. Any other use is prohibited. At a Glance Description: This is a two- day laboratory exercise in which the students will demonstrate the presence of bacteria in samples of milk. The samples differ in how they were pasteurized and the temperatures at which they have been stored and the date that they were purchased. On the first day, students will spread different milk samples on petri dishes containing nutrient agar. On the second day, colonies of bacteria will be counted and the class data averaged and analyzed. Concepts addressed in this lab include the presence of bacteria in fresh foods, the effect of temperature on bacterial growth and survival, the differences between pasteurization and sterilization, and the use of sterile technique in working with bacteria. Time Requirements: This exercise takes place over two days, but does not require a full period on either day. There is time for discussion and/or student working on questions and analysis on each day. Curriculum Placement: This exercise can be used in any life science course in conjunction with the following topics: • introduction to bacteria • microorganisms and food • scientific method • health and nutrition Equipment: To prepare materials and execute this lab you will need • an autoclave or pressure cooker • incubator at 32 degrees • hot plate to boil water Materials: 30 petri dishes (sterile, plastic) nutrient agar to make 30 petri dishes 30 sterile applicators (swabs) 7 or 8 marking pens (1 per group) 4 test tubes (<130 mm long) 4 sterile transfer pipettes 2 half pint cartons of pasteurized milk 2 half pint cartons of UHT pasteurized milk 1 bio-hazard autoclave bag (optional 2003 Science in the Real World: Microbes in Action 3 This material may be duplicated by teachers for use in the classroom. Any other use is prohibited. Time Line Prior to the lab: • teacher sterilizes tubes, makes nutrient agar and pours petri dishes • teacher buys milk at least one or more weeks prior to the lab. This becomes the "old milk". One week before lab: • Teacher sets milk containers out at room temperature (see Preparations II) One day before the lab: • teacher buys 2 fresh containers of milk • teacher sets one milk container out at room temperature The day of the lab: • Teacher boils part of the old milk sample for 5 min. • Teacher boils part of the fresh milk sample, which was left out overnight, for 5 min. • Students execute part 1 of the lab Preparation I. Materials Per Class: 4 sterile tubes 16 x 125 mm (covered) one half pint pasteurized milk as fresh as possible one half pint UHT milk 2 sterile 5 ml pipettes (sterile dropping pipettes can be substituted) 4 sterile dropping pipettes For each group of 4 students 4 petri dishes containing nutrient agar 1 marking pen to mark on petri dish 4 sterile applicators (swabs) II. Preparing Milk At least one week before the lab get a half pint of milk from the cafeteria or grocery store. Also get at least two half pints of UHT pasteurized milk. One brand name of UHT milk is Parmalat. At the beginning of each class, point out the containers to these students and tell them these will be used in a lab activity next week. Leave these containers of milk out at room temperature for the week. These cartons of milk are to demonstrate graphically to the students the fact that pasteurized milk spoils when left at room temperature, while UHT pasteurized milk does not. The students will use other cartons of milk for their experiments, and the pasteurized milk they use will have to be purchased only a day or so before the lab begins. 2003 Science in the Real World: Microbes in Action 4 This material may be duplicated by teachers for use in the classroom. Any other use is prohibited. III. Preparing Nutrient Agar When convenient, but at least one day before the lab, nutrient agar must be prepared, sterilized and the agar poured into petri dishes. In addition, test tubes (at least 4 for each class) need to be sterilized. It is easier to use culture tubes, 16 x 125 mm, and caps to hold the milk samples. Regular test tubes covered with aluminum foil will also work. Note: The tubes should be short enough for the sterile droppers to stick out of the top when they are used. The recipe for most nutrient agar is to dissolve 23 grams of nutrient agar powder into 1 liter of distilled water (tap water will work). Generally, figure on pouring 20-25 ml of nutrient agar into each dish. Prepare the agar in batches that only fill the flask half way to prevent boilover. Once you have prepared the nutrient agar, cover the flasks with aluminum foil and place them in the autoclave/pressure cooker. Also, be sure to put your sample tubes in the sterilizer at the same time (caps should be on loosely). Follow the instructions on your sterilizer to complete the sterilization process. Generally, you need 15 Ibs. of pressure and a temperature of 121º C for 15 minutes to achieve sterility. Once the materials have been sterilized, the nutrient agar must be poured into the dishes before it solidifies. It is best to pour the agar when it has cooled enough to be held comfortably in your hand (45-50º C). Spread the plates out on the lab tables, lift the lid straight up and pour the agar into the dish. You may want to pour 20 ml of water into an empty dish to give you an idea of how much agar to pour into each dish. Once all of the dishes have been poured, let them set until they have solidified. Store the plates upside down until you are going to use them. If it is going to be several days until you use the plates, put them back into the plastic sleeve in which they where shipped and store them upside down in a refrigerator or a cool place. IV. Sample Preparation The day before the lab, you need to place the samples of milk (one sample from the UHT milk and three samples from a fresh carton of pasteurized milk) into the sterile tubes. You want to work as carefully as possible to maintain sterility. For the best lab results, you should use milk that is as fresh as possible, that is, with as late an expiration date as possible. Open the carton of milk as carefully as possible. Try not to touch the opening with your fingers. Using a sterile 5 ml pipette or a sterile dropping pipette, transfer about 5 ml of the pasteurized milk into each of 3 sterile tubes (if you have 5 classes that means a total of 15 tubes). Carefully, open the UHT milk and using a new sterile 5 ml pipette, transfer 5 ml of the UHT milk into a sterile tube (again, for 5 classes you will need 5 tubes) KEEP THE TUBES COVERED AT ALL TIMES WHEN NOT MAKING A TRANSFER. For each class, place one tube of fresh pasteurized milk into the refrigerator and leave two tubes at room temperature. The tube with UHT milk will also remain at room temperature. 2003 Science in the Real World: Microbes in Action 5 This material may be duplicated by teachers for use in the classroom. Any other use is prohibited. V. Day One On the day of the lab, be sure to have the petri dishes, cotton swabs and marking pens available to the students.